Selective isomerization of retinal upon two-photon excitation

Article abstract of DOI:10.1016/S0009-2614(02)02034-1

The products of photo-isomerization when excited directly up to the two-photon-allowed ¹A_g^- excited state of alltrans and five cis isomers of retinal were determined. The composition of the isomers at the photo-stationary state was drastically changed as compared to one-photon excitation. The production of the 13-cis isomer was selectively increased, and the production of the dicis isomers was observed in the case of a direct excitation upon the 3¹A_g^- state, although they were hard to be produced by one-photon excitation in n-hexane solution. These results clearly evidenced that there exist isomerization pathways via two-photon-allowed excited states.

Full text of DOI:10.1016/S0009-2614(02)02034-1

Chemical Physics Letters 369 (2003) 380–385
www.elsevier.com/locate/cplett
Selective isomerization of retinal upon two-photon excitation
a,
b
a,c
Tokutake Sashima *, Hideki Hashimoto , Shin-ya Koshihara
a
Kanagawa Academy of Science and Technology, KSP East 301, 3-2-1 Sakado, Kawasaki 213-0012, Japan
‘Light and Control’, PRESTO/JST, Department of Physics, Graduate School of Science, Osaka City University,
3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan
b
c
Department of Materials Science, Tokyo Institute of Technology, 2-12-1 Oh-okayama, Meguro-ku, Tokyo 152-8551, Japan
Received 23 October 2002; in final form 5 December 2002
Abstract
ꢀ
1
The products of photo-isomerization when excited directly up to the two-photon-allowed Ag excited state of all-
trans and five cis isomers of retinal were determined. The composition of the isomers at the photo-stationary state was
drastically changed as compared to one-photon excitation. The production of the 13-cis isomer was selectively in-
creased, and the production of the dicis isomers was observed in the case of a direct excitation upon the 31Agꢀ state,
although they were hard to be produced by one-photon excitation in n-hexane solution. These results clearly evidenced
that there exist isomerization pathways via two-photon-allowed excited states.
Ó 2003 Elsevier Science B.V. All rights reserved.
1. Introduction
and choose a single pathway among them. This
may give us an opportunity to establish one of the
key technologies to speed up the information
transfer. Miyazawa et al. [2,3] recently reported a
highly selective photoreaction for one of the most
simple p-electron-conjugated molecules, cis-stil-
bene, in organic solvent by the use of a non-reso-
nant two-photon excitation method. One-photon
excitation of cis-stilbene produced trans-stilbene
together with dihydrophenanthrene (a cyclized
product), while non-resonant two-photon excita-
tion of it resulted in the production of only trans-
stilbene. No intermolecular cyclization occurred
in the latter case. This result suggested that there
are two pathways for the initial photoreaction of
p-electron-conjugated molecules that have two
different singlet excited states. One is from the 1Ag
Photochemical reactions of p-electron-conju-
gated molecules in vitro are more complicated
than those in vivo. A mixture of various geomet-
rical isomers is usually produced under a photo-
stationary state in vitro. On the contrary, the
selective trans ! cis or cis ! trans isomerization of
retinal, for example, takes place in bacteriorho-
dopsin (all-trans ! 13-cis) or in rhodopsin (11-
cis ! all-trans), respectively [1]. Understanding
and mimicking the way in which nature controls
these complicated photochemical reactions would
make it possible to control the reactions optically
* Corresponding author. Fax: +81-44-819-2053.
E-mail address: sashima@net.ksp.or.jp (T. Sashima).
1
state, and the other is from the Bu state.
0009-2614/03/$ - see front matter Ó 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0009-2614(02)02034-1

T. Sashima et al. / Chemical Physics Letters 369 (2003) 380–385
381
Photo-isomerization of a biologically important
p-electron-conjugated molecule, retinal, by one-
photon and/or by triplet-sensitized excitation has
been studied extensively to understand the reason
for the natural selection of a particular isomer as
well as the highly selective cis–trans isomerization
in biological systems [4,5]. A comparison between
the one-photon and triplet-sensitized excitation
revealed that the photo-isomerization of retinal
isomers in n-hexane mainly occurred via the lowest
excited triplet state [4]. On the other hand,
femtosecond time-resolved UV–VIS absorption
spectroscopy of all-trans retinal in n-hexane by
one-photon excitation showed the isomerization
pathway via the 21Ag excited state (the lowest
excited state), which is the precursor of the per-
pendicularly twisted singlet excited state [6].
In this study, we demonstrate the photo-iso-
merization of retinal isomers by direct excitation
up to a two-photon-allowed excited state and show
direct evidence for the isomerization pathway via
the 31Aꢀg stateꢀof retinal isomers by direct excita-
tion to the 31Ag state by the use of a non-resonant
two-photon excitation method.
purified using silica gel (LiChrosolb Si-60) HPLC
(4/ Â 300 mm column and 7% diethylether/
n-hexane). n-Hexane, diethyl ether, and acetoni-
trile (special grade) were purchased from Wako
Chemical Industry and used as received. The
11,13-dicis isomer was separated and purified from
the isomeric mixture obtained by the irradiation of
all-trans retinal in n-hexane with nanosecond laser
pulses of 490 nm (see below).
For photo-isomerization experiments, the con-
centration of each isomer in n-hexane solution was
adjusted to be 2 Â 10ꢀ4 M. The solutions were
irradiated with laser pulses (5 ns pulse duration;
0.8 mJ at 245 nm for one-photon excitation and
8.5 mJ at 490 nm for two-photon excitation). The
wavelength dependence for the photo-isomeriza-
tion of two-photon excitation was determined
under a fixed condition of exposure time (30 min)
and laser power (70 mW). Laser light between 440
and 820 nm was used for this purpose. All proce-
dures were performed at room temperature and
under dim red light. Isomerization without laser
irradiation was negligible. The isomeric composi-
tion after each irradiation period of time was de-
termined by HPLC, as described above.
2. Experimental
3. Results and discussion
All-trans retinal was synthesized starting from
commercially available retinyl acetate. Retinyl ac-
etate was purchased from BASF (Switzerland) and
used after purification with silica–gel column
chromatography (20% diethyl ether/n-hexane). It
was reduced with LiAlH4 and then oxidized using
activated MnO2 to produce retinal. The retinal thus
obtained was subjected to silica–gel column chro-
matography (15% diethyl ether/n-hexane), and the
all-trans component was collected. All-trans retinal
was further purified by recrystallization from an
n-hexane solution of this particular component.
Mono-cis isomers of retinal were prepared from
an isomeric mixture produced by irradiating all-
trans retinal. All-trans retinal in acetonitrile was
irradiated with a 250 W tungsten–halogen lamp
for 1 h at room temperature under a nitrogen at-
mosphere. Each isomer was separated from the
mixture of isomers by silica–gel column chroma-
tography (15% diethyl ether/n-hexane) and further
A p-electron-conjugated molecule has several
singlet excited states belonging to particular sym-
metry groups. PPP–MRD–CI calculations of
model polyenes by Tavan and Schulten [7] pre-
dicted that the energy of each singlet excited state
for the polyene with six conjugaꢀted C@C double
bonds was in the order of 31Ag > 1Buþ > 1Buꢀ >
21Aꢀg . Recently, a two-photon-allowed singlet ex-
cited state, 31Agꢀ, was newly identified for some
carotenoids by the measurements of resonance-
Raman excitation profiles [8] and electro-modula-
tion spectroscopy [9]. There is little information
concerning the energy of the optical-forbidden
singlet excited states of retinal; hence, it is sup-
posed that the ordꢀer of each singlet excited statꢀe in
n-hexane is 31Ag , 1Aþg > 1Bþu > 1Bꢀu > 21Ag >
npÃ. Note that we here adopt the notation of the
electronic states assuming the C2h symmetry of the
polyene backbone.

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T. Sashima et al. / Chemical Physics Letters 369 (2003) 380–385
The specific wavelength of the laser light to be
the 440–820 nm wavelength region, which is hardly
affected by one-photon excitation. Fig. 2shows the
results of the all-trans isomer by two-photon ex-
citation. Isomerization was not detected at all
above 540 nm excitation; however, 440–540 nm
excitations did induce isomerization. There was a
peak around 480 nm that showed the selective
production of the 13-cis isomer. A half wavelength
of that may correspond to a transition tꢀo the third
two-photon-allowed excited state ð31Ag Þ because
of its energy. Therefore, in order to fulfill the re-
quirements to satisfy the effective two-photon
excitation as well as to avoid the interference of
photoisomerization via the 1Buþ state that is in-
duced by one-photon excitation, we selected a 490
nm excitation for subsequent studies. The yield of
the cis species was proportional to the 1.9th power
of the laser power when using pulses of 490 nm.
This result shows that the isomerization by this
excitation wavelength is a two-photon reaction.
Two-photon excitation of nanosecond laser
pulses showed quite different photo-isomerization
characteristics from those of the one-photon ex-
citation and/or the triplet-sensitized excitation [4].
We tried to examine three excitation wavelengths,
i.e., 245, 355, and 490 nm. They correspond, re-
spectively, to the cis-peak (1Agþ state), the region
nearby the one-photon-allowed transition to the
1Bþu state, and a half energy of 245 nm (no ab-
sorption coefficient exists at 490 nm). A ground
used for the two-photon excitation experiment was
selected according to the following protocol. In a
previous transient absorption study of the direct
excitation upon the 21Aꢀg state using femtosecond
laser pulses [10], 800 nm excitation was applied to
two-photon excitation. Fig. 1 shows the absorp-
tion spectra of isomeric retinal in an n-hexane so-
lution at room temperature. None of the isomers
shows a one-photon-allowed transition above 440
nm. At first, we tried 800 nm excitation of nano-
second laser pulses with no success. No isomeri-
zation was observed after 2h of exposure. This
result may be due to the fact that the absorption
cross section of the two-photon-allowed electronic
ꢀ
ꢀ
1
transition ð Ag ! 21Ag Þ was small as well as the
fact that the nonlinear optical effect induced by the
femtosecond laser excitation was 3–4 orders of
magnitude larger than that induced by the nano-
second laser excitation. In order to identify the
suitable wavelengths for the two-photon excitation
using nanosecond laser pulses, we closely inspected
ꢀ
þ
ð Ag Þ to 1Ag one-photon transition is supposed to
be partially allowed even for the all-trans isomer
due to the twisting of the polyene backbone. Table
1 shows the photo-stationary state composition of
isomers excited at 245, 355, and 490 nm. Excita-
tion of 355 nm resulted in 70% all-trans and 27%
13-cis, while 245 nm excitation resulted in 71% all-
trans and 24% 13-cis. The compositions of these
isomers were almost the same for both excitations.
On the basis of the comparison of the photo-
stationary state mixture of isþomers produced by
1
1
direct excitation up to the Bu state (355 nm ex-
Fig. 1. Absorption spectra of all-trans (solid), 13-cis (dot),
11-cis (short dash), 9-cis (dash), 7-cis (dot dash), and 11,13-dicis
(2-dot dash) retinal in n-hexane. Arrows show the excitation
wavelength for one- and two-photon excitation measurements.
Inset shows the order of singlet energy levels of all-trans retinal
in n-hexane. Solid lines are one-photon allowed excited-states
and dashed lines are one-photon forbidden.
citation) and that by triplet-sensitized excitation,
Mukai et al. [4] showed that isomerization by one-
photon excitation took place via a triplet excited
state that was formed by an intersystem crossing
from the 1Bþu state. Therefore, the above result
suggests that the isomerization induced by the

T. Sashima et al. / Chemical Physics Letters 369 (2003) 380–385
383
Fig. 2. (a) The excitation-wavelength dependence on the isomeric composition for 30 min irradiation starting from all-trans retinal.
Each mark shows all-trans (s), 13-cis (}), 11-cis (O), 9-cis (Â), 7-cis (Ã), and 11,13-dicis (M). (b) The square power dependence of the
isomerization from all-trans to 13-cis retinal upon exposure of 532nm nanosecond laser pulses.
Table 1
The average value of isomeric composition starting from each retinal isomer at photo-stationary state excited at 245, 355, and 490 nm
with nanosecond laser pulse
Excitation wavelength
(nm)
Isomeric composition (%)
all-trans
13-cis
11-cis
9-cis
7-cis
11,13-dicis
245
355
490
71
70
24
24
27
42
2
0
9
2
2
6
0
0
0
0
0
18
245 nm excitation should also take place via a
triplet excited state that was fþormed through in-
of a unique isomerization pathway via the two-
photon-allowed singlet excited state. It is interest-
ing to þnote that the relaxation from the 31Agꢀ to
þ
1
1
ternal conversion from the Ag to the Bu states
followed by the intersystem crossing.
1
the Bu states does not play a major role in the
Since there is no absorption band of each reti-
nal isomer at 490 nm, this particular wavelength
should allow us to excite the two-photon-allowed
singlet excited state, i.e., the 31Agꢀ state. Even
when any retinal isomers were irradiated, the
composition of photo-stationary state isomers
upon 245 and 355 nm excitation was almost the
same value to each other; the all-trans isomer was
the main component, and the 13-cis isomer was
less than half of the all-trans isomer (see above).
On the contrary, in the case of two-photon exci-
tation at 490 nm, the 13-cis isomer was produced
as the main component, and the 11,13-dicis isomer
was also produced, although this particular isomer
was hardly detected for 245 and 355 nm excita-
tions. This result is direct evidence of the existence
isomerization induced by two-photon excitation.
The amount of 13-cis isomer under the photo-
stationary state produced by two-photon (490 nm)
excitation was obviously larger than that produced
by one-photon (245 and 355 nm) excitation. On
the other hand, preliminary experiments using
femtosecond laser pulses for two-photon excita-
tion up to the 21Agꢀ state revealed that the all-trans
and 13-cis isomers had an almost equivalent
composition at the photo-stationary state. If we
assume that isomerization takes place along the
adiabatic potential energy curves of the 31Agꢀ,
21Aꢀg , and 1Bþu states, we can explain the difference
of the isomeric composition at the photo-station-
ary state when excited up to each excited state. The
13-cis isomer was the major product when excited

384
T. Sashima et al. / Chemical Physics Letters 369 (2003) 380–385
directly up to the 31Aꢀg state. This may be due to
the fact that, on the adiabatic potential energy
curve of C13@C14 torsion, the 13-cis configura-
tion has a more stable minimum than the all-trans
configuration.
Fig. 3 shows the time course of the isomeriza-
tion excited at 245 and 490 nm starting from the
all-trans, 13-cis, 11-cis, 9-cis, 7-cis, and 11,13-dicis
isomers of retinal in n-hexane solutions. The
relative speed of isomerization for one-photon
excitation was in the order of 9-cis ꢁ 11,13-di-
cis > 11-cis > 7-cis > 13-cis ꢂ all-trans, while that
for two-photon excitation was in the order of all-
trans > 7-cis ꢁ 9-cis ꢁ 11-cis ꢁ 13-cis > 11,13-dicis.
If we compare the relative rate of isomerization at
the initial stage of one-photon and two-photon
excitations, the all-trans isomer was the slowest by
one-photon excitation but the fastest by two-
photon excitation. The 11,13-dicis isomer showed
the opposite trend of the all-trans isomer. Based on
the inference that is drawn from the above dis-
cussion concerning the different isomeric compo-
sition at photo-stationary states, the all-trans
isomer would have the most stable potential min-
imum in the one-photon-allowed excited state,
while the 11,13-dicis isomer would have it in the
two-photon-allowed excited state. This interpre-
tation does not contradict the fact that the 11,
13-dicis isomer was not detected at all by one-
photon excitation although it was substantially
produced by two-photon excitation.
4. Conclusion
In the previous photochemistry of retinal and
carotenoids, isomerization by one-photon or trip-
let-sensitized excitation was well documented,
while that through the two-photon-allowed 31Agꢀ
state was ignored in most cases. However, the
present study clearly demonstrated that this should
not always be the case. Enhancement of the pro-
duction of the 13-cis isomer was evidenced by way
of the 31Aꢀg state, and the presence of a unique
pathway that produces the 11,13-dicis isomer was
found by two-photon excitation. Since the energies
and orders of the excited states of polyene mole-
cules change systematically depending on their
Fig. 3. Time dependence of isomeric composition excited at (1)
245 and (2) 490 nm nanosecond laser pulses starting from (a)
all-trans ( ), (b) 13-cis (}), (c) 11-cis ( ), (d) 9-cis (Â), (e) 7-cis
O
ꢃ
(Ã), and (f) 11,13-dicis ( ) retinal in n-hexane.
M

T. Sashima et al. / Chemical Physics Letters 369 (2003) 380–385
385
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selective isomerization pathway and thus control
the yield of isomerization products by applying our
method of non-resonant two-photon excitation to
various biological polyene molecules. However, in
order to determine the role of 31Agꢀ excited state in
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further investigation using retinal proteins as well
as theoretical studies in terms of multi-photon
isomerization processes are necessary.
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